ALLANL. GROSSBERG A N D DAVIDPRESSMAN
5478
I a was pyrolyzed a t 100'. After 2 hours of heating, 0.02 g. (7.417,) of methyl acetate was collected. After 5 hours of heating a total of 0.03 g. ( l l . l y c ) of ester was obtained. Preparation of N,N-Diphenylaminoethyl Acetate.-Following the procedure of Pau1,'B 18.8 g. (0.088mole) of S,N-diphenylaminoethano117 and 10.2 g. (0.1 mole) of acetic anhydride were refluxed for 2 hours. The reaction mixture was then diluted with water and the pH of the solution adjusted t o 9 with sodium hydroxide solution. The alkaline solution was extracted with ether, the combined extracts washed with water and dried over sodium sulfate. Removal of the ether followed by distillation afforded 15 1 g. (67.4%) of N,N-diphenylaminoethyl acetate, b.p. 174.5175" a t 3 mm., sp. gr. 20/20 1.1120, n2*.6~1.5821; M R calcd. 74.60, found 74.31; reportedI6 b.p. 140-145' a t 1-2 mm . Anal. Calcd. for C16H17N02: C, 75.27; H, 6.71; X, 5.49. Found: C,75.40; H,6.34; N, 5.51. Pyrolysis of N,N-Diphenylaminoethyl Acetate. A. At 250" .-A 5.1-g. sample of S,N-diphenylaminoethyl acetate was heated, under a nitrogen atmosphere, a t 250" for 2 hours. No volatile products were collected. B. At 5OO0.-A 8.9-g. (0.036 mole) sample of the ester was passed through a Vycor tube packed with l / ~inch helices ( 1 6 ) P T. Paul, U.S. P a t e n t 2,401.658, June 4 , 1946 (17) Generously supplied b y the Piaugatuck Chemical Co
[COSTRIBUTION FROM
Vol. 82
and heated t o 500" by a furnace. The ester was passed through the tube a t a rate of 1.5 g. per minute. The pyrolysate, collected in a Dry Ice trap, was extracted with water and the aqueous solution diluted t o 100 ml. A . 10ml. aliquot, titrated with 0.0111 N sodium hydroxide solution using a Beckman model G pH meter, consumed 19.8 ml. of base and indicated that 0.132 g. (6.17c) of acetic acid was produced. The water-insoluble, brown oil from the pyrolysate was chromatographed on an alumina column but no K,N-diphenylvinylamine could be isolated. Pyrolysis of 2-(N-P-Hydroxyethylanilino)-4,6-dimethoxys-triazine (VII). Run No. 1.--,4 3.0-g. (0.01 mole) sample of the dimethoxy-s-triazine alcohol was pyrolyzed a t 200260" for 1.5 hours. S o volatile products were observed. The residue in the distillation flask, after recrystallization from dioxane, afforded 1.56 g. (65y6) of 2-h'-vinylanilino4-hydroxy-6-methoxy-s-triazine ( I I a ) , m.p. 254-256'. -4 mixture melting point with a sample of IIa from pyrolysis of I a was not depressed. Run No. 2.--h 20.0-g. (0.07 mole) sample of VI1 was pyrolyzed under the same conditions. The distillate, 1:) g. (43.8%), was identified as methanol, b.p. (cor.) 63.3 , ~ * O D 1.3296; reported b.p. 64.6','*& 7 2 * 0 . 5 ~ 1.3288.Isb (18) (a) W. R . G. Atkins and T. A. Wallace, J . C h e m Soc., 103, 1409 (1913); (b) F. H . Getman and V. L. Gibbons, T m o J O U R X A L , 37, 1995 (19 1.5).
DEPT. OF BIOCHEMISTRY RESEARCH, ROSWELL PARK MEMORIAL INSTITUTE, BUFFALO,S E T V
YORK ]
Nature of the Combining Site of Antibody against a Hapten Bearing a Positive Chargel BY ALLANL. GROSSBERG AND DAVIDPRESSMAN RECEIVED MARCH 3, 1960 Previous work indicated that the specific site of antibody against the positively charged phenyltrimethylamnioniuni ion contains a negative charge The present report emphasizes this and shows that the negative charge is apparently a carboxylate group since esterification ofthe antibody carboxyl groups by diazoacetamide destroys binding activity. This destruction is not due t o the esterification which occurs elsewhere than in the specific site Moroever esterification of antibody to a negative hapten does not affect its ability t o bind hapten.
One approach to the exact composition of the antibody site, e.g., which chemical groups contribute to the intimate structure of the site, is to modify the site chemically by use of reagents for reactive groups in protein. This approach has been used with varying succe:ss. 2-8 Reagents which react with only one type of residue are fewg3loand reaction conditions must be mild enough to permit retention of biological activity. Care must be taken to assure that reaction in other parts of the large antibody molecule does not obscure effects on the site or confuse the interpretation of data. Thus acetylation of antihaptens-' or a n t i ~ r o t e i n ~antibodies ,~.~ has (1) Presented a t the 136th National Meeting of the American Chemical Society, Atlantic City, s e p t . 1959. Supported in part by a grant from the National Institute of ~ ~ i e r gand y Infectious Diseases, USPHS. (2) D . Pressman and L. A. Sternberger, J . I?nmiiriul,, 66, 609 (19.51). (3) J. R . Marrack and E, S Orlans, Brit. J. Exptl. Pnthol., 36, 389 (1984).
J. Singer, Proc. Katl. A c a d . Sci U . S . , 41, 1041 (19.55). T. Katsura, J a p a n . J. Exptl. A f p d . , 26, 167 flQ55). (fi) A. Nisonoff and D. Pressman, J. Immi~iiol.,83, 138 (1959,. ( 7 ) A. F. S. A. Habeeb. H. G. Cassidy, P . Stelos and S. T. Singer,
14) S.
in)
been shown to reduce or prevent precipitation by antigen but this is due mainly to reaction with amino groups in regions elsewhere than in the combining site.6 Specific protection of the site by hapten during treatment with reagent provides a means of proving that the destructive reaction is in the site.2 It was by such methods that acetylation, when extensive, was shown to affect the combining site.6 The use of antihapten sera has a distinct advantage over the use of antiprotein sera in such studies in that information concerning the combining site of antibodies is more easilv interoreted in the former case. This is because,- unlikg antibodies against complex antigens such as proteins, the antihapten antibodies are directed against a known chemical configuration. Such systems have also been of value in indicating the presence of a charged group in the antibody site; i.e.,a positively charged group is in the site homologous for the negatively charged p-(p-azobenzene)-azobenzoate (Xi)]' and 4-azophthalate'?; also a negative charge is in the site for the positively charged @-azophenyl-
.~ I
( 8 ) K. Landsteiner, " T h e
Specificity of Serological Reactions," Harvard University Press, 1945, pp. 141-143, 262-264. (9) H . S. Olcott and H . Fraenkel-Conrat, C h e m Rius., 41, 151 (1947). (10) F. W. P u t n a m , " T h e Proteins," E d . H . Neurath and K. Bailey, Vol. I, P a r t B. Academic Press, Inc., S e w York, N . Y.,1953, pp. 893-
972.
hapten binding by dialysis equilibrium allows detection of interaction a t the site without the com(11) A. Nisonoff and D. Pressman, THISSOURNAL.7 9 , 1610 ((19.57). (12) D. Pressman and L. Pauling, ibid., 71, 2893 (1949). (13) D. Pressman, A. L. Grossberg, L. H. Pence a n d L. P a u l i n ~ , i b i d . , 68, 250 (1940).
COMBINING SITEOF ANTIBODY
Oct. 20, 1960
5479
Antisera.-Preparation and pooling of an antiserum and plications inherent in precipitation of a large polypreparation of the 7-globulin fraction from serum by the ionic antigen by the large polyionic antibody. method of KekwickzOhave been described.lg In all experiWe have now investigated the nature of the site of ments the yglobulin fraction of an antiserum pool or normal antibody produced in rabbits against the positively rabbit serum was utilized. "Anti-A," and "Anti-X," charged A, hapten. We have reduced the number refer to the ?-globulin fractions containing these antibodies, and not to specifically purified antibodies. of negative charges on the antibody by esterification respectively, Reaction of ?-Globulin with DAA.-?-Globulin solution of carboxyl groups and have looked for effects in (6 to 8% protein) was dialyzed a t 5' against 200 to 400 the site. The same reaction has also been applied volumes of 0.15 N NaC104 buffered a t pH 8 with 0.02 M to antibody against the negatively charged Xp sodium borate-boric acid buffer, to remove chloride ion which reacts with DAA. DAA was then added t o a conhapten. centration of 0.35 to 0.45 M in DAA; a drop of n-caprylic The reagent chosen was diazoacetaniide (DAA), alcohol reduced foaming. The reaction mixture was stirred which reacts with protein carboxyls under mild continuously and 0.15 N HC104 was added slowly to reach conditions and has been reported to be quite and maintain pH 5.50. The acid flow was then adjusted to maintain the pH between 5.40 and 5.65. Heating from specific; the only other group said to be attacked the magnetic stirrer motor produced a reaction temperature under the conditions employed is ~ulfhydry1.l~of 35 to 40". X o attempt was made to adjust the reaction Rabbit y-globulin appears to have 2 sulfhydryl temperature. After 1 to 1.5 hr. a duplicate amount of groups but these are masked, becoming available DAA4was added and the reaction allowed to proceed, with addition of a third portion of DXX if necessary, for reaction (with silver ion) only after denatura- subsequent until the desired degree of reaction was obtained after 2 to tion with dodecyl sulfate.15 Protein carboxyl is 4 hr. Esterification consumes hydrogen ion, allowing esterified by DAA and such treatment of anti-A, estimate of degree of reaction from the amount of acid antibody alters the antibody site itself so that i t added.z1 Following treatment, the mixture was brought to pH 7.5 and dialyzed a t 5" for 18 t o 40 hr. against pH 8cannot bind hapten. borate-buffered saline. Recovery of soluble protein varied
Experimental Methods Preparation of Haptens .-Preparation of p-aminophenyltrimethylammonium chloride, p-($-hydroxyphenylazo)phenyltrimethylammonium chloride, p-hydroxyphenyl-A, and I13l labeled p-iodobenzoic acid has been described previously.'3~'6 The preparation of I1al labeled p-iodophenyltrimethylammonium chloride was similar to that of the p-iod0~3~ benzoic acid. The unlabeled compound was prepared by methylation of p-iodoaniline with excess methyl iodide in an ethanol-water mixture containing sodium carbonate. The slightly soluble iodide salt was converted to the chloride by treatment with silver oxide and then hydrochloric acid. The chloride was crystallized from ethanol. A n d . Calcd. for CDH1&ICl: C1, 11.9. Found: C1, 11.7, 11.8. M.P. 195-197Odec. The exchange reaction was carried out a t 150-160" for 18 hr. using 3 to 5 mg. of salt and 1 to 2 mc. P. The product was isolated as the iodide and converted to the chloride. The purification process was repeated until constant specific activity was obtained. Preparations had a specific activity of about 6 mc./mM. Other haptens, or intermediates used in the above preparations, were commercial products of reagent grade. Preparation of Diazoacetamide (DAA).-Glycine methyl ester was prepared according to the method of Curtius and G0be1.I~ Methyldiazoacetate was prepared in 85 to 95% yield from the glycine methyl ester according to the method of Womack and h'elson .I8 Diazoacetamide was prepared by ammonolysis in 10 SHlOH of the methyl diazoacetate for 48 to 72 hr. a t 40". The mixture was aerated, lyophilized and then extracted with acetone. The product crystallized from acetone on cooling. Yields averaged 5 t o 870. Protein Antigens.-The immunizing antigen for anti-A, serum was made by diazotizing two portions of p-aminophenyltrimethylammonium chloride weighing 75 mg. and 150 mg. and coupling separately t o 5.00 g. portions of Bovine-y-globulin (Pentex Co. Fraction 11)in pH 9.5 borate buffer a t 0" for 30 minutes. The azo proteins were brought to PH 8.5 and dialyzed against several changes of $H 8 buffered saline, combined and made up to 1% protein, with 0.2% phenol, for inoculation. The immunizing antigen for anti-X, serum has been previously described .6Jg (14) P. E. Wilcox, Abstr. X I I t h Internl. Cong. Pure a n d Appl. Chem., New York, N. Y., 1951, pp. 60-61; as quoted in ref. 10. (15) G. Markus, personal communication, (16) M. Blau, A. C. Johnson a n d D. Pressman, Intevnat. J . Afipl. Radigtiolz and Isotopes, 3, 217 (1958). (17) Curtius and Gobel, J . prokt. Chem., 37, 169 (1888). (18) E.B. Womack and A. B. Nelson, "Organic Syntheses," Vol. 111, John Wiley and Sons, Inc., 1955, pp, 392-393. (19) A. Sisonoff and D. Pressman, J . Immunol., 80,417 (1958).
from 65 to 90%, being less a t the higher extents of reaction. Equilibrium Dialysis.-The technique as described by L'isonoff and P r e s ~ m a n 'was ~ employed, except that all samples of a single run were dialyzed against the same large volume (30 to 60 ml.) of hapten solution, to assure that the free hapten concentration of the samples was the same. Analytical Methods.-Concentration of protein was determined by optical density measurements a t 280 mp, utilizing as standard a rabbit ?-globulin solution of known nitrogen content. Concentration of the colored hapten, p-hydroxyphenyl-il,, was determined by optical density measurements a t 445 mp, utilizing a standard curve and correcting for effect of ?-globulin solution on the optical density. Concentration of radioactive haptens was determined in a well-type scintillation counter, standardized daily with solutions of known concentration. A minimum of 10,000 counts was recorded for each sample, corresponding to a maximum probable counting error (corrected for background) of 1.2%. Concentration of standard solutions of labeled haptens was determined through use of standard for curves of optical density us. concentration-at 238 p-iodophenyltrimethylammonium chloride and a t 248 mM for p-iodobenzoate. Titration of untreated and esterified -,-globulin was performed as described by Alexander and Cousens.2z Carboxyl groups were estimated by the acid consumed in the range pH 2 to 5.8. Determinations were reproducible to i 2% for a single protein preparation. An average value of 110 f 5 titratable carboxyl groups per 160,000 gm. protein was found for several different samples of rabbit 7globulin from normal serum or antiserum. The total number of free carboxyl groups in rabbit yglobulin estimated from amino acid analyses has been reportedz3 to be 121. DXA-treated samples always assayed as having carboxyl groups below 9.5 per 160,000 g. protein. Since the determination indicated variation of rt 5% in titratable carboxyl content of different samples of untreated 7-globulin, percentage esterification of a DAA-treated sample was calculated on the basis of the carboxyl content of its control untreated sample exposed to exactly the same procedures of dialysis and assay.
Results Effect of DAA-Treatment of Anti-A, on Hapten Binding.-The binding of haptens by anti-A, was reduced following treatment of the antibody by DAA, as shown in Table I, in which the binding by (20) R. A. Kekwick, Biochem. J., 34, 1248 (1940). (21) M.S. Doscher a n d P . E. Wilcox, Fed. Proc., IS, 217 (1959). (22) P. Alexander and S. F. Cousens, Biochem. Pharmacol., 1, 25 (1958).
(23) E. L. Smith, M. C. McFadden, A. Stockell and V. BuettnerJanusch, J . B i d . Chem., 214, 197 (1955).
ALLANL.
5480
CROSSBERG AND
TABLE I EFFECTOF DAA TREATMENT ON THE BINDING CAPACITY OF ANTI-A, ANTIBODY y-Globulin preparation Hapten 1 I"l p iodophenyltrimethylammoniud
2
#-HydroxyphenylAPd
Sample" Untreated D A A treated S o r m a l ygloburin
Carboxyl esterified (%)
Hapten conc. hound ( X 10' M ) b 0 33.9 1 0 2 14 3z 2 18 0 =t . 2
0 0.86h Untreated 0 21.1 DAAtreated 12 f 2 1 1 . 2 -f Normal yglobulin 0 2.9 f
*
.03
DAVID PKESSM.%N
TABLE I1 PROTECTION OF A N T I - A ~ANTIBODYBY HAPTENS AGAINST ACTIONOF DAA Car-~
Expt. no.
the treated globulins is compared with that of the untreated controls. Two different haptens, labeled 9-iodophenyltrimethylammonium ion and p-hydroxyphenyl-A,, were used and the same effect was observed for each. Protection of Anti-A, by Hapten During Treatment with DAA.-Anti-A, was treated with DAA in the presence of hapten to determine if the reduced hapten binding demonstrated by DAAtreated anti-A, was due to an attack on the antibody site itself or on other parts of the molecule. A separate sample of anti-A, was treated with DAA in a parallel manner with no hapten present. Assay revealed that samples esterified with and without hapten (antibody "protected" or "unprotected") were esterified to approximately the same extent (Table 11). Following dialysis to remove excess hapten, binding measurements were made on the protected and unprotected samples and on untreated anti-A,. The results (Table 11) reveal that the presence of hapten during DAA treatment reduced the effect of this treatment on hapten binding. Thus, in a preparation esterified to 13%, 9hydroxyphenyl-A, a t 5 X lo-* M prevented completely the 47% reduction in binding shown by the unprotected DAA treated anti-A,. In the other anti-A, preparations, in which esterification was more intense, with a correspondingly greater reduction in binding by the unprotected samples, protection by phenyltrimethylammonium ion and by labeled p-iodophenyltrimethylammonium ion was readily apparent, although incomplete. DAA Treatment of Anti-X,.-DAA treatment did not affect the binding of labeled p-iodobenzoate by anti-X, (Experiment 1, Table 111). To assure that conditions of DAA-treatment of anti-A, and anti-X, were comparable, mixtures of the two antibody-containing y-globulin fractions were treated. Binding measurements on these treated mixtures, compared to those of the untreated mixtures (Table 111, Experiments 2 and 3), show that the binding of hapten by DAA-treated anti-A, was markedly reduced-to 37 and 33%, respectively, of the binding shown by the untreated mixture. In contrast, binding by DAA-treated antiX, in the mixtures was not reduced. The increase in hapten binding of about 15% by DAA-treated anti-X, is also shown by DAA-treated normal rabbit y-globulin (Table 111, Experiment 4). Binding
DAA treatment
Hapten present
l b
2=
Concn. A, hapten bound ( X 106 M)'
boxyl esterified
(Yo)
None Treated
0 12
21.1 3 ~ 0 . 5 11.2 f .8
Treated
13
21.1 1 .1
None None None Treated Trimethylphenylammonium chloride (1 X A[) Treated
0 27
3 5 . 5 f .05 1 1 . 7 f .05
24
24.2 f .05
h'one None p-H ydroxyphen ylA,-(5 X lO-'M)
.5 .8 .2
Binding measurements performed a t protein concentration of 20 mg./ml. Average of duplicate determinations with average deviation; binding by antibody corrected for the binding by norma! y-globulin. Free hapten concenM. Free hapten concentration: tration: 7.19 X 4.04 X 10-6 M .
Vol. 82
None None 0 32.24 f .05 Treated 18 13.74 f .02 None p-Iodophenyltrimethylammonium chloride (5 X M ) Treated 18 26.03 f .25 Average of duplicate determinations with average deviation; values corrected for binding by normal yglobulin. Protein concentration, 20 mg./ml. 9-Hydroxyphenyl-A, used for binding measurements; free I1allabeled p-iodophenylconcentration: 4.04 X 10-6 M . triniethylammonium chloride used for binding mensurements; free concentration: experiment 2, 7.45 X 10-6 M ; experiment 3,5.75 X 10-6 M . 3O
TABLE I11
EFFECT OF DAA TREATMENT ON THE HAPTEN BINDINGOF MIXTURES OF ANTI-A,
AND
ANTI-X, ANTIBODIES Concn. A,
Experiment
Cnrhoxyl esterified Simple4
(%)
hap-cen bound ( X 105
Concn. 1'3' h-iodobkzoate hound 6 ( X 106
M)b
Anti-X, untreated 0 6.63 1" Anti-X,, DAA treated 17 f 2 6.70 Normal 7-globulin 0.19 Anti-X, Anti-A,, untreated 0 35.5 5.00 2d Anti-X, Anti-Ap, DAA treated 27 f 3 11.7 5.80 0 2.30 0.64 Normal 7-globulin Anti-X, Anti-A,, un0 26.4 6.20 treated 3" Anti-Xp Anti-A,, DAA treated 24 1 3 9 . 7 7.05 Xormal y-globulin 0 3.70 0.35 Normal 7-globulin, un0 0.64' 0.37 treated 4' Normal 7-globulin, DAA treated 30 1 2 0.40' 0.91 a Binding measurements performed a t protein concentration of 20 mg./ml. * Average of duplicate determinations with average deviation of f 3y0; binding by antibody corrected for binding by normal ?-globulin. e Free hapten concentration: 3.71 x 10- M . A, hapten: labeled p-iodophenyltrimethylammonium chloride; free concentration: 7.45 X 10-6 M ; free 1'31 labeled p-iodobenzoate concentration: 3.21 X 10-6 M . "A, hapten: p-hydroxyphenyl-A,; free concentration, 4.30 X 10-6 M ; freo 11*' labeled p-iodobenzoate concentration, 4.32 X 10-6 M . f A, hapten as in experiment 2; free concentration, 5.75 X 10-6 M ; free 1131 labeled p-iodobenzoate concentration, Measured with another preparation. 4.67 X 10- M .
COMBINING SITEOF ANTIBODY
Oct. 20, 1960
5481
100.
80
u
OO
2
4
6
I
~ (FREE HAPTEN) x 1 6MOLAR. Fig. 1.-Binding of I'31 labeled p-iodophenyltrimethylammonium ion by DAA-treated and untreated Anti-A, antibody a t 5 O , with the antibody preparation of experiment 1,Table I.
of I l 3 l labeled p-iodophenyltrimethylammonium ion by normal y-globulin was not appreciably affected by DAA-treatment of the protein. Combining Constants of DAA-treated and Untreated Anti-A,.-The extent of the binding of 113' labeled p-iodophenyltrimethylammonium ion at several levels of free hapten concentration was determined with untreated anti-A, and with antiA, esterified to 160J0. The binding results are plotted in Fig. 1 as the reciprocal of the free hapten concentrations against the reciprocals of the bound hapten concentrations. As previously discussedls the ordinate intercept of such a plot provides an estimate of the reciprocal of the total number of binding sites present in the sample; the reciprocal of free hapten concentration a t which half of these total number of sites are occupied gives an estimate of KO'the average combining constant for the sites in the sample. From Fig. 2, it was calculated that the concentration of combining sites in the 20 mg./ ml. protein solutions is 41.5 X 10+ M for the untreated anti-A,, and 24.1 X 10" M for the DAA treated anti-A,, corresponding to destruction of 42% of the sites. The KO'values are 8.7 X lo6 and 5.6 X lo5, respectively, showing that esterification reduced the binding strength of the remaining sites by 35%. Dependence of Destruction of Sites on the Exof tent of Esterification of Anti-A,.-Results binding measurements on all anti-A, samples treated with DAA are plotted in Fig. 2 as percentage reduction in binding (binding by the untreated samples taken as 100%) on a logarithmic scale, against per cent esterification of carboxyl. The destruction of binding sites progresses as more protein carboxyl is esterified. Hydrolysis of DAA Treated Anti-A,.-DAAtreated and untreated anti-A, samples were hydrolyzed under two sets of conditions of p H and temperature to determine if hydrolysis of ester linkages would lead to recovery of binding capacity. The pertinent details and the data on binding measurements are included in Table IV. Hydrolysis of ester groups was accompanied by about 20%. recovery of the binding capacity lost by the original
I-
\
301
't
1 1 1 1 1 IO
2o0
Oh
20
30
40
CARBOXYL ESTERIFIED
Fig. 2.-The effect of esterification of antibody protein carboxyl on the binding capacity of Anti-A, antibody; diameter of a point represents the estimated reliability of the extent of esterification.
DAA treatment. The hydrolysis procedures did not cause loss of binding capacity in the untreated anti-A,. TABLE IV EFFECTOF HYDROLYSIS OF DAA-TREATED AND UNTREATED ANTI-A, ANTIBODYO O N HAPTENBINDINGCAPACITIES DAA treatment
Hydrolysisb
Carboxyl content (groups/ 160,000 9.)
Conc. hapten bound ( M X 106)c
None None 101 40. Od Treated None 85 21.3d None pH11.0; 5' 105 41.7 f 0 . 5 Treated 112 25.4 f .6 , p H 9 . T ; 25' 4 0 . 5 f .2 None Treated 115 24.6 f .4 a The samples used were aliquots of the untreated and DAA treated Anti--4, proteins of experiment 1, Table I. 30 mg. of protein samples in 1.2 ml. were dialyzed for 18hr. against 500 ml. of borate buffer under the conditions indicated. 20 mg./ml. of protein concentration. Hapten: Ilal labeled p-iodophenyltrimethylammonium chloride; free concentration, 14.5 X 10- M ; values are averages of duplicate determinations, with average deviation indicated, corrected for binding by normal 7-globulin; binding was measured a t pH 8.0. Calculated from the curves of Fig. 2 for binding a t the free hapten concentration used. e Not measured.
Evidence that DAA Alkylates Only Carboxyls.Rabbit y-globulin was esterified to 30% by DAA and tested to determine whether any alkylation of other reactive groups occurred. No decrease in the amino content was detectable by van Slyke
nitrogen determination, 2 4 indicating that less than an estimated 3 to 4% of the amino groups reacted. S o shift in the tyrosine light absorption curve was observed, indicating that less than an estimated .3y0of the tyrosines were alkylated.22 KO decrease in the degree of coupling of diazotized sulfanilic acid was found, suggesting that there was also little alkylation of imidazole by DAA. Titration of free sulfhydrylz6indicated that one of the two masked sulfhydryls had been alkylated. Discussion Previous reports,6a13showed that anti-A, antibody appears to have a negative charge in the site which accommodates the positive charge of the antigen and that anti-X, antibody formed against a negative charge has a positive charge in the site. The present work emphasizes the difference in the antibody combining sites of the anti-i-l, and antiX, antibodies. When a mixture of these antibodies is treated with D M , with resultant esterification of carboxylate, only the anti-& antibody is affected, although the anti-X, was esterified simultaneously in the s a n e solution. The extent of esterification must have been identical for both antibodies. This is proof that a carboxylate or a t least a moiety capable of reacting with DAA is present in the anti-A, site, while no such grouping is present in the anti-benzoate site. That it is the attack on the site itself and not the non-specific (24) 1.P. Peters and D. D. Van Slyke, “Quantitative Clinical Chemistry,” T h e XVilliams and \\’ilkins Co., Baltimore, h f d . , 1932, PII. :i8fi399. (‘25) Kindly performed by Dr. G. Markus by the metllod reported h y G LIarkus and F.Karrish, T r ~ r sJ O U R X A L , 79, 134 (1957).
alteration of the anti-A, nlolecule which is responsible for the effect is further emphasized by the fact that esterification of anti-A, antibody in the presence of the specific hapten results in a retention of antibody activity. Specific protection would not be obtained unless the attack were on the antibody site. That esterification is responsible for the loss of activity is indicated by the fact that binding activity can be partially recovered by exposing the altered antibody to pH 11, which hydrolyzes the ester groups. These findings provide clear-cut evidence, implied by previous work,11-13 that the specific sites in antibodies against different antigens have different amino acid composition. This does not differentiate between involvement of different parts of the same polypeptide or the associated tertiary structure or to variations in the sequence of amino acids in the chain a t the region of the sites. From the rate of decrease of binding activity with esterification-70yo loss of activity when 30yo of the carboxyls are esterified-it would appear that the carboxyl in the site is more easily esterified than some of the other carboxyls. Part of this loss of binding activity may be due to a non-specific effect as shown by the fact that when 16% of the carboxyls are esterified there is a 3570 decrease in the value of &’. This non-specific effect may be partially electrostatic due to the increased positive charge of the antibody as a whole, resulting in decreased attraction or increased repulsion of the positive hapten. Steric effects due to an ester group close to the antibody site and distortion of the molecule as a whole may also contribute to the decrease in binding constant.
[COYTKIBUTIOS FROM THE DEPARTRIEST O F CHEMISTRY, U N I V E R S I T Y O F \VISCONSIN, M A D I S O S , \ v I S C O N S I N ]
The pH Dependence of the Competitive Inhibition of Fumarase l,’ BY PAUL W. ~ V I G L E RAND ~ ROBERT A. XLBERTY RECEIVED APRIL 1, 1960 Iii order to obtaiii further iiiforination on the structure and fuiiction of the active site of fumarase, the inliibitur dissociation constants with four competitive inhibitors, succinate, D-tartrate, L-tartrate and nzeso-tartrate, were determined over :L range of PH values between j>H 3.3 and 8.3. The disociation constants of the inhibitor-enzyme compounds were determined using plots of (.Y),’t wvsus (1)a t each pl-I. From these data, the two acid dissociation constants (K*EIand KI,EI) for each of the inhibitor-enzyme compounds and the dissrxiation constants of the inhibitor-enzyme conipounds of the three ionized fomms of the enzymatic site were calculated. It was fmmd that the Kazr and K h E I values for the D and I,-tartrRte compounds of fumarase are loiver than the corresponding values for the succinate compound, while the values for the n~esotartrate compound are higher. Thc K a and ~ K~h E [ values are related to the fact that the hydroxyl groups of D and 1,tartrate are in the cis configuration while the hydroxyl group’ of mcso-tartrate are in the trans configuration when the carboxylate groups are Imns. The results are interpreted in tcrms of a model for the active site based on a tmns drhydratiwi (Jf r,-rnalate by fumarase.
Introduction The divalext anions of several dicarboxylic acids are competitive ixhibitors of pig heart fumarase. (1) T h i s research was supported by grants from t h e National Science Foundation and from the Research Committee of t h e Graduate School of the Univrrsity of Wisconsin from fund, supplied by t h e Wisconsin Alumni Researcli Foundation. (2) Presented in p a r t before the 61st Annual Meeting of the American Society of Biological Chemists, Chicago, April 11-13, 1960. (3) This investigation was carried o u t during the tenure of a N ational Institutes of Health Postdoctoral Research Fellowship from the Xational Heart Institutc. (4) V. Masscy, Aiocheru . I . , 56, 172 (1’35.3)
111 order to further elucidate the relatioiisliips between the spatial configuration of the inhibitur and the e n z p ~ a t i csite, experiments with the three isomers of tartrate and with succinate were carried out over a range of pH. It has been demonstrated that there are two acidic groups which have a total effect on the kinetics of fumarase.6 The pH dependence of the inhibitor dissociation constant ( K I ) may be interpreted in terms of this model of the enzymatic site.’ The 1 . 5 ) C. l;ried?n, Ph.1). ’Thesis, UnirersiLy of Wisctinsin, 19.75, p. 76. (6) C . liriedcii u n d R . A. Alherty, J . Rid. C‘henz., 212, 859 (1955).
